Novel approach to design, development and characterization of Itraconazole bilosomes for solubility enhancement

Itraconazole is an FDA-approved antifungal drug that is poorly soluble (1). There are numerous antifungal formulations of this medication available in the market. But there is need to boost solubility of poorly soluble Itraconazole drug for greater penetration in the skin. Because bile salts are present in bilosomes-a novel modified version of liposomes and neosomes. Itraconazole is a type of antifungal drug which are used to treat different types of infections caused by fungus (2). Itraconazole is composed of imidazole. This drug has a limited rate of absorption when taken orally. Itraconazole is class II drug with low permeability. Topical medication administration is preferred to combat this since it stops the drugs first- pass metabolism (10).

 Bilosomes have the advantage of increased absorption and penetration. Drugs that are poorly soluble and strongly lipophilic are delivered by bilosomes. Bilosomes are more efficient than neosomes and liposomes. (4).  There are many formulations of neosomes and liposomes of various drugs in market but bilosomal formulation contains bile salts like sodium deoxycholate or sodium glycocholate and surfactants which are helpful for better penetration (13). So bilosomes are preferred to deliver poorly soluble drugs. Bilosomes contain a variety of bile salts, such as sodium glycocholate and sodium deoxycholate, because of their amphiphilic nature. The outer layer of bilosomes includes bile salts and hydrophobic drugs, whereas the core layer contains hydrophilic drugs. (13).  The drug Itraconazole is effective against a variety of fungus and has fewer adverse effects. However, Itraconazole has a low solubility and penetrating power. The solubility of Itraconazole in water is poor. The purpose of this work was to produce bilosomes, to increase the permeability of Itraconazole topically (15).

Evaluation of bilosomes:

Appearance: Visual observation was done to test the appearance of all 8 batches of bilosomes.

Determination of pH: pH meter was used to check the pH of the all 8 batches. The pH was measured  at 1, 30, 60, 90 days to detect if there is any change in pH as per time passes.

Vesicle size: Instrument used is Zetasizer. Sample was prepared by adding 0.1 ml bilosomes and dilute it with 100 time’s double distilled water.  The diluted sample was placed in a cuvette and then it was analysed, at temperature 25°C and scattering angle of 90° or 174° C.

Zeta potential: Sample was prepared by adding 0.1 ml bilosomes and dilute it with 10 ml deionized water.  Then it is filtered through 0.02 µm membrane for accuracy.  Zeta Potential Analyser was set at 25°C. Clean electrophoresis cell and Smoluchowski model was used because it is standard for bilosomes. And then zeta potential was calculated.

Flexibility index: It was calculated by passing the bilosomes through membrane having pores smaller than their diameter. Firsly need to measure initial particle size of bilosomes and then pass the bilosomes in membrane with lower pore size with pressure and then measure the final particle size of bilosomes after passing through this membrane.

Flexibility Index = vesicle size after extrusion   x  100

                                      Membrane pore size

 Homogenicity testing: Visual observation was done to test the homoenicity of all 8 batches of bilosomes.

Entrapment efficiency: Firstly add 1 ml bilosomes in centrifuge tube and allow to rotate at 20000-40000 rpm for 1 hour at 4° C. and then we have removed the supernatant liquid and the remaining liquid is analysed by UV visible spectrophotometer at 262nm.We have calculated amount of dug using calibration curve.

Entrapment efficiency = Total drug-free drug     x100

                                                       Total drug

 Sun exposure testing: While performing sun exposure evaluation bilosomes were placed in sunlight for the time period of  24 hours and after 24 hours the chnages in bilosomes were observed by the visual observation.

 Freeze thaw testing: While performing freeze thaw testing bilosomes were placed in freeze at low temperature and then bilosomes were taken from freeze and placed at room temperature. We need to repeat this cycle for 5 times and then we had observed changes by visual observation of all 8 batches.

 Drug release testing: The drug diffusion test of all 8 batches of bilosomes were done by Franz diffusion Cellophane membrane was used. Franz diffusion cell is made up of 2 compartments. In Franz diffusion cell one compartment is donor and other compartment  is receiver. Cellophane mebrane was placed in between these 2 compartments.Bilosomes were placed on cellophane membrane and drug release was calculated for total period of 6 hours.

 Drug content: 1 ml bilosomes were dissolved in 30 ml methanol. Then stir I continuously for 1 hour. Sonicate the sample. Taken the absorbance of the sample at 262nm and then drug content was calculated.

Stability study: All 8 batches were kept at room temperature for stability study.

 Antifungal activity: F4 batch was optimised batch out of 8 batches of bilosomes due to high drug content and high drug release. We have performed antifungal study of F4 batch. Minimum Inhibitory Concentration of drug was calculated firstly against fungus Candida albicans. Agar disc-diffusion assay method was used for antifungal study. Zones of inhibitions were measured and compared with the marketed formulation of drug and pure drug also.

UV visible spectrum of Itraconazole

By UV visible spectroscopic analysis maximum wavelength of Itraconazole was found to be 255nm.

Fig 1. Uultra violet visible spectrum of Itraconazole drug

Table 2: Absorbance range of Itraconazole at different concentration

                                             Fig 2.Calibration curve of Itraconazole

IR Spectroscopy:

Fig 3.  IR spectra of Itraconazole drug

XRD Analysis of Itraconazole drug

                                   Fig.4. XRD Graph of Itraconazole

                                 Fig.5. Itraconazole peak mark XRD graph

Flexibility Index, Entrapment Efficiency, Drug Release Testing, Drug content:

Table 3: Flexibility Index, Entrapment Efficiency, Drug Release Testing, Drug content of all batches

Zeta Potential: Zeta Potential of optimised batch 4 was calculated and it was found to be 28 mv.

Homogenicity Testing: All batches of bilosomes were homogenous by visual observation.

Sun Exposure Testing: In the sun exposure evaluation bilosomes does not show any changes as per  visual observation.

Freeze Thaw Testing: In freeze thaw testing bilosomes does not show any changes as per  visual observation.

Stability study: All formulations are stable for 3 months at room temperature

Antifungal Activity: MIC of the drug against the Candida albicans was calculated and it was found to be 3.6 ug/ml. And then zones of inhibitions were calculated for drug, bilosomes and marketed formulation of drug.

Table 4: Zone of inhibition of different sample

Fig.6. Zone of inhibition of Itraconazole drug, bilosomes and marketed formulation in Candida albicans cultur

Bilosomes of Itraconazole were prepared and evaluated by using sodium deoxyglycolate, span 60 and cholesterol. F4 batch shows higher drug diffusion and higher drug content, so F4 batch was concluded as optimised batch. Itraconazole bilosomes increases drug solubility and penetration capacity of drug in skin. Itraconazole is a poorly souble drug and here we had increased solubility of this drug by forming a novel dosage form bilosomes. Hence this study concluded that F4 was optimized batch which was stable for 3 months and had a higher drug diffusion rate.

1.      Hashem S.M. Itraconazole-Loaded Ufasomes: Evaluation, Characterization, and Anti-Fungal Activity against Candida albicans, Pharmaceutics, 2022, 15( 1). 1-20,

2.      Lamie C. Assessment of antifungal efficacy of itraconazole loaded aspasomal cream:comparative clinical study, Drug Delivery, 2022, 29( 1). 1345–1357.

3.      Usman F. Itraconazole Loaded Biosurfactin Micelles with Enhanced Antifungal Activity: Fabrication, Evaluation and Molecular Simulation, Antibiotics, 2023 12( 1). 1-15.

4.      Yoshida I.. Intravenous itraconazole compared with liposomal amphotericin B as empirical antifungal therapy in patients with neutropaenia and persistent fever, Mycoses, 2020 63(1), 794–801.

5.      Adel S. Fabrication and optimization of itraconazole-loaded zein-based nanoparticles in coated capsules as a promising colon-targeting approach pursuing opportunistic fungal infections, Drug Delivery and Translational Research, 2023, 13 (1) 2982–3002.

6.      Poojari C.Cholesterol Reduces Partitioning of Antifungal Drug Itraconazole into Lipid Bilayers, The Journal of Physical Chemistry, 2020, 124(1) 2139−2148,

7.      Alyahya E.M. Preparation and Optimization of

8.      Itraconazole Transferosomes- Loaded HPMC Hydrogel for Enhancing Its Antifungal Activity: 2ˆ3 Full Factorial Design, Polymers, 2023, 15 (4), 995

9.      Kim J.  Itraconazole, a Commonly Used Antifungal that Inhibits Hedgehog Pathway Activity and Cancer Growth, Cancer Cell, 2023 17(4)388–399,

10.   Adel A. A. Itraconazole, a Commonly Used Antifungal that Inhibits Hedgehog Pathway Activity and Cancer Growth, Cancer Cell, 2019, 161 (4),87-95

11.   Abdelhakeem E. Revitalizing Itraconazole: Unleashing its Anticancer Potential through Oral Nanosystems for Liver Targeting and Biodistribution Profiling in an Animal Model using Radiolabeling Technique, Journal of Drug Delivery Science and Technology, 2024, 104 (1), 106463

12.   Wagh V. Itraconazole Niosomes Drug Delivery System and Its Antimycotic Activity against Candida albicans, ISRN Pharmaceutics, 2021, 2012(1), 1-7

13.   Mahajan H., Serum and sebum pharmacokinetics evaluation of a novel formulation of itraconazole in healthy volunteers, Indian Journal of Drugs in Dermatology, 2022,8(1), 7-14

14.   Khudhair A. Preparation and evaluation of topical hydrogel containing ketoconazole-loaded bilosomes, Acta Pharmaceutica Sciencia, 2024, 62(4), 762-777

15.   Rukari T. View of Vesicular drug delivery systems for the fungal infections’ treatment through topical application-a systemic review, Journal of current science and technology, 2023 13(2) 501-513

16.   Saito A. Efficacy of Intravenous Itraconazole Versus Liposomal Amphotericin B as Empirical Antifungal Therapy in Hematological Malignancy with Persistent Fever and Neutropenia: Study Protocol for a Multicenter, Prospective, Randomized Non- inferiority Trial Edited by tatsuyuki kakuma Study Protocol, Kurume medical journal, 2019 8(2)

17.   . 501-513

18.   Pace J., Formulation and evaluation of itraconazole liposomes for Hedgehog pathway inhibition, Journal of Liposome Research, 2019,30(3) 305- 311.

19.   Spec AMSG-15: Super-Bioavailability Itraconazole Versus Conventional Itraconazole in the Treatment of Endemic Mycoses - A Multicenter, Open-Label, Randomized Comparative Trial, Open Forum Infectious Diseases,2024, 11(3) 1- 8.

20.   Rukari T. Vesicular drug delivery systems for the fungal infections’ treatment through topical application-a systemic review, Journal of Current Science and Technology, 2023 13(2).501-517

21.   Harousseau J. Itraconazole Oral Solution for Primary Prophylaxis of Fungal Infections in Patients with Hematological Malignancy and Profound Neutropenia: a Randomized, Double-Blind, Double-Placebo, Multicenter Trial Comparing Itraconazole and Amphotericin B, antimicrobial agents and chemotherapy, 2020 44(7) 1887-1893.

22.   Sampathi S. preparation and evaluation of liposome entrapped hydrogel complex systems of itraconazole for enhanced transdermal permeation, Journal of Pharmaceutical & Scientific Innovation,2014 (2014), 3(1). 25-29.

23.   Curic A. Formulation optimization of itraconazole loaded PEGylated liposomes for parenteral administration by using design of experiments, International Journal of Pharmaceutics, 2013, 448(1) 189-197

24.   Wang J. Preparation of itraconazole-loaded liposomes coated by carboxymethyl chitosan and its pharmacokinetics and tissue distribution, Drug Delivery, 2011 18(8). 631-638.

25.   Kumar N. Development, characterization and preclinical evaluation of nanosized liposomes of itraconazole for topical application: full factorial design to estimate the relationship between formulation components, Journal of Drug Delivery Science and Technology,2021 66(1) 1-9.

26.   Leal A. Antifungal Activity of a Liposomal Itraconazole Formulation in Experimental Aspergillus flavus Keratitis with Endophthalmitis, Mycopathologia, 2015 179(4) 225-229.